A HR-MS Based Method for the Determination of Chorismate Synthase Activity.

Chorismate synthase (Cs) catalyzes the last step of Shikimate pathway involving a unique biochemical reaction of anti-1,4 elimination of 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate-3-phosphate (EPSP) leading to the formation of chorismate, which is the common precursor for aromatic amino acid, ubiquinone, and folate biosynthesis in plants and several bacterial, fungal, and parasitic pathogens. Absence of Shikimate pathway in the vertebrate host, make Cs an appealing target for drug discovery against these pathogens. Here, we report a new method for detection of chorismate through a specific liquid chromatography, coupled with negative electrospray ionization high-resolution tandem mass spectrometry (ESI-HRMS) for determination of Cs enzyme activity. For this, we used a coupled enzyme reaction consisting of purified recombinant MtbEPSPs (EPSP synthase from Mycobacterium tuberculosis) for biosynthesis of EPSP, which is the substrate for Chorismate synthase along with MtbCs (Chorismate synthase both from Mycobacterium tuberculosis) for the formation of chorismate, followed by its detection through LC/HRMS. Since, the reaction components of Cs enzyme activity assay which otherwise may interfere with the other known spectrophotometric methods of checking Cs enzyme activity have no effect on this LC/HRMS based method, this method offer advantages over other existing methods for detection of Cs activity. Further, this LC/HRMS based method could be applicable for detection of enzyme activity of both monofunctional and bifunctional Cs from different species irrespective of their specific requirements of anaerobic or aerobic reaction conditions.

Diguanylate cyclase activation: it takes two.

Characterization of an activated diguanylate cyclase reported in this issue of Structure by Wassmann et al. (2007) reveals how phosphorylation promotes dimerization necessary for synthesis of the second messenger c-di-GMP, establishes the catalytic mechanism, and identifies a widely conserved mode of product inhibition.

Structure of BeF3- -modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition.

Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger involved in the regulation of cell surface-associated traits and persistence. We have determined the crystal structure of PleD from Caulobacter crescentus, a response regulator with a diguanylate cyclase (DGC) domain, in its activated form. The BeF(3)(-) modification of its receiver domain causes rearrangement with respect to an adaptor domain, which, in turn, promotes dimer formation, allowing for the efficient encounter of two symmetric catalytic domains. The substrate analog GTPalphaS and two putative cations are bound to the active sites in a manner similar to adenylate cyclases, suggesting an analogous two-metal catalytic mechanism. An allosteric c-di-GMP-binding mode that crosslinks DGC and an adaptor domain had been identified before. Here, a second mode is observed that crosslinks the DGC domains within a PleD dimer. Both modes cause noncompetitive product inhibition by domain immobilization.

Nuclear localization of tetrahydrobiopterin biosynthetic enzymes.

Biosynthesis of the tetrahydrobiopterin (BH(4)) cofactor, essential for catecholamines and serotonin production and nitric oxide synthase (NOS) activity, requires the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). Upon studying the distribution of GTPCH and PTPS with polyclonal immune sera in cross sections of rat brain, prominent nuclear staining in many neurons was observed besides strong staining in peri-ventricular structures. Furthermore, localization studies in transgenic mice expressing a Pts-LacZ gene fusion containing the N-terminal 35 amino acids of PTPS revealed beta-galactosidase in the nucleus of neurons. In contrast, PTPS-beta-galactosidase was exclusively cytoplasmic in the convoluted kidney tubules but nuclear in other parts of the nephron, indicating again that nuclear targeting may occur only in specific cell categories. Furthermore, the N terminus of PTPS acts as a domain able to target the PTPS-beta-galactosidase fusion protein to the nucleus. In transiently transfected COS-1 cells, which do not express GTPCH and PTPS endogenously, we found cytoplasmic and nuclear staining for GTPCH and PTPS. To further investigate nuclear localization of all three BH(4)-biosynthetic enzymes, we expressed Flag-fusion proteins in transiently transfected COS-1 cells and analyzed the distribution by immunolocalization and sub-cellular fractionation using anti-Flag antibodies and enzymatic assays. Whereas 5-10% of total GTPCH and PTPS and approximately 1% of total SR were present in the nucleus, only GTPCH was confirmed to be an active enzyme in nuclear fractions. The in vitro studies together with the tissue staining corroborate specific nuclear localization of BH(4)-biosynthetic proteins with yet unknown biological function.

Mechanism of chorismate synthase. Role of the two invariant histidine residues in the active site.

Chorismate synthase catalyzes the last step in the common shikimate pathway leading to aromatic compounds such as the aromatic amino acids. The reaction consists of the 1,4-anti-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate. Although this reaction does not involve a net redox change, the enzyme has an absolute requirement for reduced flavin mononucleotide, which is not consumed during the reaction. Two invariant histidine residues are found in the active site of the enzyme: His(17) and His(106). Using site-directed mutagenesis, both histidines were replaced by alanine, reducing the activity 10- and 20-fold in the H106A and H17A mutant protein, respectively. Based on the characterization of the two single mutant proteins, it is proposed that His(106) serves to protonate the monoanionic reduced FMN, whereas His(17) protonates the leaving phosphate group of the substrate. An enzymatic reaction mechanism in keeping with the experimental results is presented.

Identification of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in human DNA polymerase lambda: a possible role in base excision repair.

Base excision repair (BER) is a major repair pathway in eukaryotic cells responsible for repair of lesions that give rise to abasic (AP) sites in DNA. Pivotal to this process is the 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity of DNA polymerase beta (Pol beta). DNA polymerase lambda (Pol lambda) is a recently identified eukaryotic DNA polymerase that is homologous to Pol beta. We show here that human Pol lambda exhibits dRP lyase, but not AP lyase, activity in vitro and that this activity is consistent with a beta-elimination mechanism. Accordingly, a single amino acid substitution (K310A) eliminated more than 90% of the wild-type dRP lyase activity, thus suggesting that Lys(310) of Pol lambda is the main nucleophile involved in the reaction. The dRP lyase activity of Pol lambda, in coordination with its polymerization activity, efficiently repaired uracil-containing DNA in an in vitro reconstituted BER reaction. These results suggest that Pol lambda may participate in "single-nucleotide" base excision repair in mammalian cells.

Chorismate synthase from the hyperthermophile Thermotoga maritima combines thermostability and increased rigidity with catalytic and spectral properties similar to mesophilic counterparts.

Chorismate synthase, the last enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate, a biochemically unique reaction in that it requires reduced FMN as a cofactor. Here we report on the cloning, expression, and characterization of the protein for the first time from an extremophilic organism Thermotoga maritima which is also one of the oldest and most slowly evolving eubacteria. The protein is monofunctional in that it does not have an intrinsic ability to reduce the FMN cofactor and thereby reflecting the nature of the ancestral enzyme. Circular dichroism studies indicate that the melting temperature of the T. maritima protein is above 92 degrees C compared with 54 degrees C for the homologous Escherichia coli protein while analytical ultracentrifugation showed that both proteins have the same quaternary structure. Interestingly, UV-visible spectral studies revealed that the dissociation constants for both oxidized FMN and 5-enolpyruvylshikimate 3-phosphate decrease 46- and 10-fold, respectively, upon heat treatment of the T. maritima protein. The heat treatment also results in the trapping of the flavin cofactor in an apolar environment, a feature which is enhanced by the presence of the substrate 5-enolpyruvylshikimate 3-phosphate. Nevertheless, stopped-flow spectrophotometric evidence suggests that the mechanism of the T. maritima protein is similar to that of the E. coli protein. In essence, the study shows that T. maritima chorismate synthase exhibits considerably higher rigidity and thermostability while it has conserved features relevant to its catalytic function.